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28 results for “hdac11”
HDAC11 target engagement assay development in cells - CETSA
<p><strong>Methods</strong></p> <p><strong>Permeabilized CETSA</strong></p> <p>HEK293T cells were seeded in 6-well plates (8 x 10<sup>5</sup> cells/well) in DMEM supplemented with 10% FBS , penicillin (100 U/mL) and streptomycin (100 µg/mL). 4 h after seeding cells were transfected with 0.2 µg of N-terminally NanoLuc-tagged HDAC11 and 1.8 µg of empty pcDNA3.1 vectors using X-tremeGENE™ HP transfection reagent, following manufacturer instructions. Next day cells were harvested using Trypsin-EDTA, and resuspended in pre-warmed Opti-MEM (no phenol-red) at a concentration of 1.2 x 10<sup>5</sup> cells/ml. A 10x stock solution of digitonin (500 µg/ml, Sigma) and 100x stock solution of protease inhibitor cocktail (cOmplete™, Mini, EDTA-free,<em> </em>Sigma<em>)</em> was prepared in Opti-MEM and DMSO, respectively. The protease inhibitor / DMSO stock solution was used as vehicle to prepare 100x stock solutions of all test compounds. The 10x digitonin stock solution and 100x test compound / vehicle stock solution were pre-dispensed into 96-well PCR reaction plates at 10 µl/well and 1 µl/well, respectively. Next, 89 µl/well of the cell suspension was added to the pre-loaded PCR plates. Plates were then covered with adhesive plate sealing tape and were incubated at 37°C for 1 h. Immediately prior to heating the PCR plates, a 5x substrate stock solution was prepared by diluting NanoBRET NanoGlo Substrate (Promega) 1:100 into Opti-MEM. The thermal cycler was allowed to reach the designated temperature prior to placement of the PCR plate into the heating block. For Tm determination experiments, duplicate plates containing a fixed concentration of test compounds (30 µM) as well as vehicle control were prepared to perform a 11 point temperature gradient spanning 37-68.5°C for 3 minutes. The plates were allowed to cool at room temperature for 3 minutes after the heat treatment followed by addition of 25 µl 5x substrate stock solution per well. To measure NanoLuc activity 100 µl of sample were transferred from the PCR plate to a white 96-well plate. Total luminescence was measured immediately after substrate addition with CLARIOstar microplate reader (BMG Labtech).</p> <p><strong>Non-permeabilized CETSA</strong></p> <p>HEK293T cells were seeded in 6 well plates (8 x 10<sup>5</sup> cells/well) in DMEM supplemented with 10% FBS , penicillin (100 U/mL) and streptomycin (100 µg/mL). 4 h after seeding cells were transfected with 0.2 µg of N-terminally NanoLuc tagged HDAC11 and 1.8 µg of empty pcDNA3.1 vectors using X-tremeGENE™ HP transfection reagent, following manufacturer instructions. Next day cells were tretaed with compounds/DMSO for 6 h. Cells were harvested using Trypsin-EDTA and resuspended in pre-warmed Opti-MEM (no phenol-red) at a concentration of 1 x 10<sup>5</sup> cells/ml. Next, 100 µl/well of the cell suspension was added to the pre-loaded PCR plates. Immediately prior to heating the PCR plates, a 5x substrate stock solution was prepared by diluting NanoBRET NanoGlo Substrate (Promega) 1:100 into Opti-MEM. The thermal cycler was allowed to reach the designated temperature prior to placement of the PCR plate into the heating block. For Tm determination experiments, duplicate plates containing a fixed concentration of test compounds (30 µM) as well as vehicle control were prepared to perform a 11 point temperature gradient spanning 37-72°C for 3 minutes. The plates were allowed to cool at room temperature for 3 minutes after the heat treatment followed by addition of 25 µl 5x substrate stock solution per well. To measure NanoLuc activity 100 µl of sample were transferred from the PCR plate to a white 96-well plate. Total luminescence was measured immediately after substrate addition with CLARIOstar microplate reader (BMG Labtech).</p> <p> </p>
The process of HDAC11 Assay Development: using kit
<p>Preliminary tests to check if the Fluor-de-Lys-green HDAC activity kit could be used for developing a high-throughput HDAC11 assay have been performed here using controls and recombinantly purified HDAC11.</p>
The process of HDAC11 Assay Development: enzyme stability
<p>Before performing the kinetic study for calculating the Km for HDAC11, it is important to know the duration of the stability of the protein under the assay conditions. This is being analyzed here.</p> <p> </p> <p><strong>Note: </strong>1. In the assay buffer, BSA conc. is 0.5 mg/ml (instead of 0.5%).</p> <p> 2. In the 7.5 ul developer solution, 40 uM of TSA (Trichostatin A) is also included.</p>
The process of HDAC11 Assay Development: buffer screening
<p>A buffer screen at various pH was performed to pick the buffer that gives the best activity for HDAC11. </p> <p>Note: 1. In the assay buffer, BSA conc. is 0.5 mg/ml (instead of 0.5%).</p> <p> 2. In the 7.5 ul developer solution, 40 uM of TSA (Trichostatin A) is also included.</p>
The process of HDAC11 Assay Development: linearity and Km calculation
<p>With previously optimized conditions, the linearity range for HDAC11 was obtained and the Km for the substrate was calculated.</p> <p> </p> <p><strong>Note: </strong>1. In the assay buffer, BSA conc. is 0.5 mg/ml (instead of 0.5%).</p> <p> 2. In the 7.5 ul developer solution, 40 uM of TSA (Trichostatin A) is also included.</p>
The process of HDAC11 Assay Development: Buffer optimization again
<p>The pH range used earlier was expanded for further optimization to check if the activity could improve.</p> <p> </p> <p><strong>Note: </strong>1. In the assay buffer, BSA conc. is 0.5 mg/ml (instead of 0.5%).</p> <p> 2. In the 7.5 ul developer solution, 40 uM of TSA (Trichostatin A) is also included.</p>
The process of HDAC11 Assay Development: Effect of DMSO
<p>For the purpose of compound library screenings, the compounds would be made available in DMSO and thus, when they would be incubated with the protein, the protein solution will now contain DMSO as well. It is important to determine the concentration of DMSO that could be tolerated by the protein such that the protein stays active. Hence, the effect of varying concentration of DMSO was analyzed on HDAC11 activity.</p>
The process of HDAC11 Assay Development: Effect of Detergent
<p>The presence of detergent can prevent the sticking of the fluorophore to the sides of the wells of the plate and thus improve the signals. Hence, the effect of detergent was observed on the overall signals and the activity of HDAC11.</p>
The process of HDAC11 Assay Development: Check for linearity and Km-repeat
<p>The linearity for HDAC11 activity for Boc-Lys-(TFA)-AMC substrate was monitored using the newly optimized buffer conditions. This was followed upon by Km calculation.</p>
The process of HDAC11 Assay Development: time-course study follow-up-effect of additives
<p>Since the activity of HDAC11 has been observed to be quite low as per the previous data, a preliminary test was performed to check any drastic increase in the activity in the presence of an additive. </p> <p>version2 (v2) has updated reaction recipe.</p>
The process of HDAC11 Assay Development: time-course study follow-up
<p>The time of incubation and conc. of the developer to be used are important parameters during HDAC11 assay optimization and thus, have been considered here.</p> <p>Version2 (v2) has updated reaction recipe.</p>
The process of HDAC11 Assay Development: time-course study follow-up
<p>A follow-up to check the increased fluorescence signals at 0 min of the HDAC11 reaction with its substrate was performed. </p> <p>Version2 (v2) is updated with the reaction recipe.</p>
The process of HDAC11 Assay Development: time-course study
<p>Using Boc-Lys-(TFA)-AMC substrate, a preliminary time-course study was initiated to determine the appropriate HDAC11 concentration and the time to be used for the reaction (which are essential parameters to perform Km calculations later).</p> <p>The updated version2 (v2) has the corrected reaction recipe.</p>
The process of HDAC11 Assay Development: a kit-free method
<p>In order to develop an assay for HDAC11, a kit-free method is being attempted wherein Boc-Lys-(TFA)-AMC, a previously reported substrate was used to check the activity of HDAC11 in a preliminary experiment.</p> <p>In version2 (v2), the reaction recipe components are corrected (a missing component in previous version).</p>
The process of HDAC11 Assay Development: Z'-factor
<p>As a last step, to check if the assay qualifies for the screening purposes, Z’-factor was calculated here.</p>
HDAC11 protein purification
<p>Recombinant human HDAC11 protein was purified.</p>
The process of HDAC11 Assay Development: Effect of reducing agent
<p>An effect of adding reducing agent on the HDAC11 activity was monitored.</p>
HDAC11 cellular assay development- histones deacetylation
<p>I looked at the effect of overexpression of HDAC11 on exogenous histone acetylation in HEK293T cells.</p>
Lactate production by Staphylococcus aureus biofilm inhibits HDAC11 to reprogramme the host immune response during persistent infection [RNA-seq]
GEO Series GSE135495. Mus musculus. 2 samples. Type: Expression profiling by high throughput sequencing.
E0-12h_HDAC11-495
GEO Series GSE25964. Drosophila melanogaster. 6 samples. Type: Genome binding/occupancy profiling by genome tiling array.
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